Polypeptide-human SNARE protein 25 and a polynucleotide encoding the same

ABSTRACT

The present invention discloses a novel polypeptide-Human SNARE protein 25 and polynucleotide encoding the same, as well as a method of producing the polypeptide by DNA recombinant technology. The present invention also discloses methods of using the polypeptide in treatment of various diseases, such as malignant tumor, blood disease, HIV infection, immunological disease and various inflammations. The present invention also discloses an antagonist against the polypeptide and the therapeutic use of the same. The present invention also discloses the use of such polynucleotide encoding Human SNARE protein 25.

FIELD OF INVENTION

The invention relates to the field of biotechnology. In particular, theinvention relates to a novel polypeptide, Human SNARE protein 25, and apolynucleotide sequence encoding said polypeptide. The invention alsorelates to the method for the preparation and use of said polynucleotideand polypeptide.

TECHNICAL BACKGROUND

Plasma membrane plays an important role in cell life because allconnections and interactions between cells and the environment must passthrough the membrane. For instance, substances go into or out of a cellthrough the plasma membrane. Hormones and drugs act on cells via plasmamembrane, and metabolism regulation, cell recognition and immunization,etc, all are related to and depend on the functions of plasma membrane.

Substances get into or out of a cell via plasma membrane. In live cells,plasma membrane is highly selective with regard to substancepermeability. There are three major ways for substances to pass throughplasma membrane: passive transportation, active transportation, andendocytosis and exocytosis.

Protein transportation in eukaryotes includes secretion, endocytosis,and exocytosis (Nature 1994 Nov. 3; 372(6501): 55-63).

Macromolecules, such as proteins, polynucleotides and polysaccharidescan only pass through plasma membrane via endocytosis and exocytosis.The mechanism of transporting these macromolecules is different fromthat of transporting small molecules such as solutes and ions, andinvolves the orderly formation and fusion of small vesicles along theplasma membrane. Endocytosis and exocytosis are both activetransportation, because they need energy supply. One important featureof endocytosis and exocytosis is that the macromolecules are enclosed inthe vesicle rather than being mixed up with other macromolecules ororganelles. This ensures that macromolecules outside or inside themembrane can be orderly transported. Rapid vesicular formation andfusion in large numbers are one of the basic traits of all eukaryoticcells.

Cells have complex internal membrane systems. A transporting vesicleobtains its contents from the donor organelle selectively, and fuseswith the membrane in a highly selective manner as well. Accordingly, allvesicles must have surface markers which allow the recognition of thetarget membrane based on their sources and contents, and the targetmembrane must also have corresponding receptors. Though the mechanism ofthis recognition remains to be elucidated, a widely noticed theory isthat the recognition involves proteins called SNAREs (“SNAP Receptors”).v-SNARE present on the vesicle membrane and t-SNARE present on targetmembrane have complementary structure and directs vesiculartransportation. During the process, a transporting vesicle may examinemany possible targets on the membranes before its v-SNARE finds thecomplementary t-SNARE. According to this theory the recognition isregulated by members of an enzyme family called Rab protein. Rabproteins examine whether the pairing between v-SNARE and t-SNARE iscorrect. Rab protein binds to the surface of the coated vesicle whilethe donor membrane is budding to form the vesicles. When the vesiclemeets the target membrane, v-SNARE couples with SNARE for a sufficientperiod of time to allow Rab protein to hydrolyze GTP, and to anchor thevesicle on the target membrane, and subsequent fusion.

The fusion of the inner membranes is catalyzed by a specific fusionprotein, which can overcome the inherent energy barrier. Little wasknown about the mechanism, other than that the process needs ATP, GTP,acyl-coenzyme A and several other proteins, two of which are known: NSF(N-ethylmaleimide-sensitive fusion protein) and SNAPs (soluble NSFattachment proteins). SNAPs shuttle between the fusing membrane andcytoplasma, and bind to v-SNARE on the vesicle membrane and t-SNARE onthe target membrane, initiating the assembly of the fusion machinery,which catalyzes the fusion of the lipid bilayers.

SNARE is a family of proteins. The basic functions of SNARE include: (1)it plays a significant regulatory role in the recognition betweenvesicular and target membranes; and (2) it catalyzes the fusion of lipidbilayers. Besides, because NSF greatly influences the secretory processof platelets and neurotransmitter releases, SNARE, as receptors of NSF,is also important in those processes. (Blood. 1999 94: 1313-8; J.Neurosci. 1998 18: 10241-9). Therefore SNARE plays important roles inthe transportation of macromolecules such as proteins, transportation ina cell, the influence of hormone and drugs on cells, metabolismregulation, cell recognition and immunity etc.

The Golgi complex is known to be involved in exocytosis. Some secretoryproteins exit the cell through the Golgi complex. The Golgi complex isalso a main site of carbohydrate synthesis, where glycoproteins andpolysaccharides are synthesized. Aminopolysaccharides are also sulfatedin the Golgi complex.

The Golgi complex is also a place for sorting and delivering theproducts of ER. A large portion of carbohydrates produced in the Golgicomplex are attached to proteins and fatty acids from ER asoligosaccharide side chains. Some oligosaccheride groups function asmarkers and direct proteins to lysosomes or other cellular compartments.

Two types of SNARE have been found in the Golgi complex. Their molecularweight is 28KD and 27KD, respectively, and in humans are located onchromosomes 17q11 and 17q21. They contain a coiled-coil functionaldomain in the center and an anchoring site on the carboxyl terminal(Science 1996, 272: 1161-3).

In Vitro tests indicate that SNARE protein plays an important role inthe recognition and fusion of vesicles and target membrane during thecellular transportation between ER and Golgi complex. (Science 1996,272: 1161-3; J. Cell Biol. 1996, 133: 507-16)

When a vesicle touches the target membrane, SNARE integrates withsoluble SNAP and NSF and forms a 20s complex which promotes membranefusion. NSF acts as ATPase, probably supplying energy to overcome theenergy barrier (Mammalian Genome 7: 850-852, 1996).N-ethylmaleimide-sensitive factor (NSF) is an ATPase which is related tovesicular fusion in eukaryotes. NSF together with SNAP decomposecis-SNARE complex through hydrolyzing ATP, leading to the formation oftrans-SNARE complex. Therefore SNARE is important in the forming ofproteins in ER-Golgi complex and some relevant transporting processes(Mol Cell 199 Jul. 4(1): 97-107).

Gene mapping data place human GS27 near the gene which causes hereditaryhypertension, and there are suggestions that the gene encoding GS27 maybe helpful in the diagnoses and treatment of hereditary hypertension(Genomics 1999; 57(2): 285-8).

Through amino acid sequence comparison, the present inventors identifieda new human SNARE protein 25 (hSNARE25). It is homologous to SNARE-29KDin rat Golgi complex (database accession # AF035823). hSNARE25 isbelieved to have some biological functions as SNARE-29KD.

As mentioned above hSNARE25 plays an important role in the regulation ofcell division and embryo development. Moreover the regulation process isbelieved to involve many proteins, so there is a need to identify moreproteins involved in these processes, especially to identify their aminoacid sequences. The isolation of the gene which codes for SNARE25protein also forms a foundation for identifying the protein's function,both under healthy and pathologic conditions, and for developingdiagnostic and treatment methods.

DESCRIPTION OF THE INVENTION

One objective of the invention is to provide an isolated novelpolypeptide, i.e., a Human SNARE protein 25, and fragments, analoguesand derivatives thereof.

Another objective of the invention is to provide a polynucleotideencoding said polypeptide.

Another objective of the invention is to provide a recombinant vectorcontaining a polynucleotide encoding a Human SNARE protein 25.

Another objective of the invention is to provide a geneticallyengineered host cell containing a polynucleotide encoding a Human SNAREprotein 25.

Another objective of the invention is to provide a method for producinga Human SNARE protein 25.

Another objective of the invention is to provide an antibody against aHuman SNARE protein 25 of the invention.

Another objective of the invention is to provide mimetics, antagonists,agonists, and inhibitors for the polypeptide of the Human SNARE protein25.

Another objective of the invention is to provide a method for thediagnosis and treatment of the diseases associated with an abnormalityof Human SNARE protein 25.

The present invention relates to an isolated polypeptide, which isoriginated from human, and comprises a polypeptide having the amino acidsequence of SEQ ID NO: 2, or its conservative variants, or its activefragments, or its active derivatives and its analogues. Preferably, thepolypeptide has the amino acid sequence of SEQ ID NO: 2.

The present invention also relates to an isolated polynucleotide,comprising a nucleotide sequence or its variant selected from the groupconsisting of (a) the polynucleotide encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2; and (b) a polynucleotidecomplementary to the polynucleotide (a); (c) a polynucleotide thatshares at least 92% homology to the polynucleotide (a) or (b).Preferably, said nucleotide sequence is selected from the groupconsisting of (a) the sequence of position 6172-846 in SEQ ID NO: 1; and(b) the sequence of position 1-4201 in SEQ ID NO: 1.

The invention also includes: a vector containing a polynucleotide ofsaid invention, especially an expression vector; a host cell geneticallyengineered with the vector via transformation, transduction ortransfection; a method for the production of said inventive polypeptidethrough the process of host cell cultivation and expression productharvest.

The invention also relates to an antibody which specifically binds tothe inventive polypeptide.

The invention also relates to a method for selecting compounds whichcould simulate, activate, antagonize, or inhibit the activity of theinventive polypeptide and the compounds obtained by the method.

The invention also relates to a method for in vitro diagnosis method ofthe diseases or disease susceptibility related with the abnormalexpression of the inventive polypeptide. The method involves thedetection of mutation in the polypeptide or its encoding polynucleotidesequence, or the determination of its quantity and/or biologicalactivity in biological samples.

The invention also relates to pharmaceutical compositions whichcomprises the inventive polypeptide, its analogues, mimetics, agonists,antagonists, inhibitors, and a pharmaceutically acceptable carrier.

The invention also relates to applications of the inventive polypeptideand/or its polynucleotide for drug development to treat cancers,developmental diseases, immune diseases, or other diseases caused byabnormal expression of the inventive polypeptide.

Other aspects of the invention are apparent to the skilled in the art inview of the disclosure set forth hereinbelow.

The terms used in this specification and claims have the followingmeanings, unless otherwise noted.

“Nucleotide sequence” refers to oligonucleotide, nucleotide, orpolynucleotide and parts of polynucleotide. It also refers to genomic orsynthetic DNA or RNA, which could be single stranded or double stranded,and could represent the sense strand or the antisense strand. Similarly,the term “amino acid sequence” refers to oligopeptide, peptide,polypeptide, or protein sequence and parts of proteins. When the “aminoacid sequence” in the invention is related to the sequence of a naturalprotein, the amino acid sequence of said “peptide” or “protein” will notbe limited to be identical to the sequence of that natural protein.

“Variant” of a protein or polynucleotide refers to the amino acidsequence or nucleotide sequence, respectively with one or more aminoacids or one or more nucleotides changed. Such changes include deletion,insertion, and/or substitution of amino acids in the amino acidsequence, or of nucleotides in the polynucleotide sequence. Thesechanges could be conservative and the substituted amino acid has similarstructural or chemical characteristics as the original one, such as thesubstitution of Ile with Leu. Changes also could be not conservative,such as the substitution of Ala with Trp.

“Deletion” refers to the deletion of one or several amino acids in theamino acid sequence, or of one or several nucleotides in the nucleotidesequence.

“Insertion” or “addition” refers to the addition of one or several aminoacids in the amino acid sequence, or of one or several nucleotides inthe nucleotide sequence, comparing to the natural molecule.“Substitution” refers to the change of one or several amino acids, or ofone or several nucleotides, into different ones without changing numberof the residues.

“Biological activity” refers to structural, regulatory or biochemicalcharacteristics of a natural molecule. Similarly, the term“immungenecity” refers to the ability of natural, recombinant, orsynthetic proteins to inducing a specific immunological reaction, or ofbinding specific antibody in appropriate kind of animal or cell.

“Agonist” refers to molecules which regulate, but generally enhance theactivity of the inventive polypeptide by binding and changing it.Agonists include proteins, nucleotides, carbohydrates or any othermolecules which could bind the inventive polypeptide.

“Antagonist” or “inhibitor” refers to molecules which inhibit or downregulate the biological activity or immunogenecity the inventivepolypeptide via binding to it. Antagonists or inhibitors includeproteins, nucleotides, carbohydrates or any other molecules which bindto the inventive polypeptide.

“Regulation” refers to changes in function of the inventive polypeptide,including up-regulation or down-regulation of the protein activity,changes in binding specificity, changes of any other biologicalcharacteristics, functional or immune characteristics.

“Substantially pure” refers to the condition of substantially free ofother naturally related proteins, lipids, saccharides, or othersubstances. One of ordinary skill in the art can purify the inventivepolypeptide by standard protein purification techniques. Substantiallypure polypeptide of the invention produces a single main band in adenaturing polyacrylamide gel. The purity of a polypeptide may also beanalyzed by amino acid sequence analysis.

“Complementary” or “complementation” refers to the binding ofpolynucleotides by base pairing under the condition of approximate ionconditions and temperature. For instance, the sequence “C-T-G-A” couldbind its complementary sequence “G-A-C-T.” The complementation betweentwo single strand molecules could be partial or complete. Homology orsequence similarity between two single strands obviously influences theefficiency and strength of the formed hybrid.

“Homology” refers to the complementary degree, which may be partially orcompletely homologous. “Partial homology” refers to a sequence beingpartially complementary to a target nucleotide. The sequence could atleast partially inhibit the hybridization between a completelycomplementary sequence and the target nucleotide. Inhibition ofhybridization could be assayed by hybridization (Southern blot orNorthern blot) under less stringent conditions. Substantiallycomplementary sequence or hybrid probe could compete with the completelycomplementary sequence and inhibit its hybridization with the targetsequence under less stringent conditions. This doesn't mean thatnonspecific binding is allowed under a less stringent condition, becausespecific or selective reaction is still required.

“Sequence Identity” refers to the percentage of sequence identity orsimilarity when two or several amino acid or nucleotide sequences arecompared. Sequence identity may be determined by computer programs suchas MEGALIGN (Lasergene Software Package, DNASTAR, Inc., Madison Wis.).MEGALIGN can compare two or several sequences using differentmethodologies such as the Cluster method (Higgins, D. G. and P. M. Sharp(1988) Gene 73:237-244). Cluster method examines the distance betweenall pairs and arrange the sequences into clusters. Then the clusters arepartitioned by pair or group. The sequence identity between two aminoacid sequences such as sequence A and B can be calculated by thefollowing equation: $\frac{\begin{matrix}{{Number}\quad{of}\quad{paired}\quad{identical}\quad{residues}} \\{{between}\quad{sequences}\quad A\quad{and}\quad B}\end{matrix}}{\begin{matrix}{{{Residue}\quad{number}\quad{of}\quad{sequence}\quad A} -} \\{{{number}\quad{of}\quad{gap}\quad{residues}\quad{in}\quad{sequence}\quad A} -} \\{{number}\quad{of}\quad{gap}\quad{residue}\quad{in}\quad{sequence}\quad B}\end{matrix}} \times 100$

Sequence identity between nucleotide sequences can also be determined byCluster method or other well-known methods in the art such as the JotunHein method (Hein J., (1990) Methods in Enzymology 183:625-645)

“Similarity” refers to the degree of identity or conservativesubstitution degree of amino acid residues in corresponding sites of theamino acid sequences when compared to each other. Amino acids forconservative substitution are: negative charged amino acids includingAsp and Glu; positive charged amino acids including Leu, Ile and Val;Gly and Ala; Asn and Gln; Ser and Thr; Phe and Tyr.

“Antisense” refers to the nucleotide sequences complementary to aspecific DNA or RNA sequence. “Antisense strand” refers to thenucleotide strand complementary to the “sense strand.”

“Derivative” refers to the inventive polypeptide or the chemicallymodified nucleotide encoding it. This kind of modified chemical can bederived from replacement of the hydrogen atom with Alkyl, Acyl, orAmino. The nucleotide derivative can encode peptide retaining the majorbiological characteristics of the natural molecule.

“Antibody” refers to the intact antibody or its fragments such as Fa,F(ab′)2 and Fv, and it can specifically bind to antigenic epitopes ofthe inventive polypeptide.

“Humanized antibody” refers to an antibody which has its amino acidsequence in non-antigen binding region replaced to mimic human antibodyand still retain the original binding activity.

The term “isolated” refers to the removal of a material out of itsoriginal environment (for instance, if it's naturally produced, originalenvironment refers to its natural environment). For example, a naturallyproduced polynucleotide or a polypeptide in its original host organismmeans it has not been “isolated,” while the separation of thepolynucleotide or a polypeptide from its coexisting materials in naturalsystem means it was “isolated.” This polynucleotide may be a part of avector, or a part of a compound. Since the vector or compound is notpart of its natural environment, the polynucleotide or peptide is still“isolated.”

As used herein, the term “isolated” refers to a substance which has beenisolated from the original environment. For naturally occurringsubstance, the original environment is the natural environment. Forexample, the polynucleotide and polypeptide in a naturally occurringstate in the viable cells are not isolated or purified. However, if thesame polynucleotide and polypeptide have been isolated from othercomponents naturally accompanying them, they are isolated or purified.

As used herein, “isolated human SNARE protein 25,” means that humanSNARE protein 25 does not essentially contain other proteins, lipids,carbohydrate or any other substances associated therewith in nature. Theskilled in the art can purify human SNARE protein 25, by standardprotein purification techniques. The purified polypeptide forms a singlemain band on a non-reducing PAGE gel. The purity of human SNARE protein25 can also be analyzed by amino acid sequence analysis.

The invention provides a novel polypeptide—human SNARE protein 25, whichcomprises the amino acid sequence shown in SEQ ID NO: 2. The polypeptideof the invention may be a recombinant polypeptide, natural polypeptide,or synthetic polypeptide, preferably a recombinant polypeptide. Thepolypeptide of the invention may be a purified natural product or achemically synthetic product. Alternatively, it may be produced fromprokaryotic or eukaryotic hosts, such as bacterial, yeast, higher plant,insect, and mammal cells, using recombinant techniques. Depending on thehost used in the protocol of recombinant production, the polypeptide ofthe invention may be glycosylated or non-glycosylated. The polypeptideof the invention may or may not comprise the starting Met residue.

The invention further comprises fragments, derivatives and analogues ofhuman SNARE protein 25. As used in the invention, the terms “fragment,”“derivative” and “analogue” mean the polypeptide that essentiallyretains the same biological functions or activity of human SNARE protein25 of the invention. The fragment, derivative or analogue of thepolypeptide of the invention may be (i) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code; or (ii) one in which one or more of the amino acidresidues are substituted with other residues, including a substituentgroup; or (iii) one in which the mature polypeptide is fused withanother compound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol); or (iv) one in whichadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthe skilled in the art from the teachings herein.

The invention provides an isolated nucleic acid or polynucleotide whichcomprises the polynucleotide encoding an amino acid sequence of SEQ IDNO: 2. The polynucleotide sequence of the invention includes thenucleotide sequence of SEQ ID NO: 1. The polynucleotide of the inventionwas identified in a human embryonic brain cDNA library. Preferably, itcomprises a full-length polynucleotide sequence of 4201 bp, whose ORF(172-846) encodes 224 amino acids. Based on amino acid homologycomparison, it is found that the encoded polypeptide is 91% homologousto SNARE-29KD in rat Golgi complex. This novel human SNARE protein 25has similar structures and biological functions to those of SNARE-29KDin rat Golgi complex.

The polynucleotide according to the invention may be in the forms of DNAor RNA. The forms of DNA include cDNA, genomic DNA, and synthetic DNA,etc., in single stranded or double stranded form. DNA may be an encodingstrand or a non-encoding strand. The coding sequence for maturepolypeptide may be identical to the coding sequence shown in SEQ ID NO:1, or is a degenerate sequence. As used herein, the term “degeneratesequence” means a sequence which encodes a protein or peptide comprisinga sequence of SEQ ID NO: 2 and which has a nucleotide sequence differentfrom the sequence of coding region in SEQ ID NO: 1.

The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2includes those encoding only the mature polypeptide, those encodingmature polypeptide plus various additional coding sequence, the codingsequence for mature polypeptide (and optional additional encodingsequence) plus the non-coding sequence.

The term “polynucleotide encoding the polypeptide” includespolynucleotides encoding said polypeptide and polynucleotides comprisingadditional coding and/or non-coding sequences.

The invention further relates to variants of the above polynucleotideswhich encode a polypeptide having the same amino acid sequence ofinvention, or a fragment, analogue and derivative of said polypeptide.The variant of the polynucleotide may be a naturally occurring allelicvariant or a non-naturally occurring variant. Such nucleotide variantsinclude substitution, deletion, and insertion variants. As known in theart, an allelic variant may have a substitution, deletion, and insertionof one or more nucleotides without substantially changing the functionsof the encoded polypeptide.

The present invention further relates to polynucleotides, whichhybridize to the hereinabove-described sequences, that is, there is atleast 50% and preferably at least 70% identity between the sequences.The present invention particularly relates to polynucleotides, whichhybridize to the polynucleotides of the invention under stringentconditions. As herein used, the term “stringent conditions” means thefollowing conditions: (1) hybridization and washing under low ionicstrength and high temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2)hybridization after adding denaturants, such as 50% (v/v) formamide,0.1% bovine serum/0.1% Ficoll, 42° C.; or (3) hybridization only whenthe homology of two sequences at least 95%, preferably 97%. Further, thepolynucleotides which hybridize to the hereinabove describedpolynucleotides encode a polypeptide which retains the same biologicalfunction and activity as the mature polypeptide of SEQ ID NO: 2

The invention also relates to nucleic acid fragments hybridized with thehereinabove sequence. As used in the present invention, the length ofthe “nucleic acid fragment” is at least more than 10 bp, preferably atleast 20-30 bp, more preferably at least 50-60 bp, and most preferablyat least 100 bp. The nucleic acid fragment can be used in amplificationtechniques of nucleic acid, such as PCR, so as to determine and/orisolate the polynucleotide encoding human SNARE protein 25.

The polypeptide and polynucleotide of the invention are preferably inthe isolated form, preferably purified to be homogenous.

According to the invention, the specific nucleic acid sequence encodinghuman SNARE protein 25 can be obtained in various ways. For example, thepolynucleotide is isolated by hybridization techniques well-known in theart, which include, but are not limited to 1) the hybridization betweena probe and genomic or cDNA library so as to select a homologouspolynucleotid6 sequence, and 2) antibody screening of expression libraryso as to obtain polynucleotide fragments encoding polypeptides havingcommon structural features.

According to the invention, DNA fragment sequences may further beobtained by the following methods: 1) isolating double-stranded DNAsequence from genomic DNA; and 2) chemical synthesis of DNA sequence soas to obtain the double-stranded DNA.

Among the above methods, the isolation of genomic DNA is leastfrequently used. A commonly used method is the direct chemical synthesisof DNA sequence. A more frequently used method is the isolation of cDNAsequence. Standard methods for isolating the cDNA of interest is toisolate mRNA from donor cells that highly express said gene followed byreverse transcription of mRNA to form plasmid or phage cDNA library.There are many established techniques for extracting mRNA and the kitsare commercially available (e.g. Qiagene). Conventional method can beused to construct cDNA library (Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). ThecDNA libraries are also commercially available. For example, ClontechLtd. has various cDNA libraries. When PCR is further used, even anextremely small amount of expression products can be cloned.

Numerous well-known methods can be used for screening for thepolynucleotide of the invention from cDNA library. These methodsinclude, but are not limited to, (1) DNA-DNA or DNA-RNA hybridization;(2) the appearance or loss of the function of the marker-gene; (3) thedetermination of the level of human SNARE protein 25 transcripts; (4)the determination of protein product of gene expression by immunologymethods or the biological activity assays. The above methods can be usedalone or in combination.

In method (1), the probe used in the hybridization could be homologousto any portion of polynucleotide of invention. The length of probe istypically at least 10 nucleotides, preferably at least 30 nucleotides,more preferably at least 50 nucleotides, and most preferably at least100 nucleotides. Furthermore, the length of the probe is usually lessthan 2000 nucleotides, preferably less than 1000 nucleotides. The probeusually is the DNA sequence chemically synthesized on the basis of thesequence information. Of course, the gene of the invention itself or itsfragment can be used as a probe. The labels for DNA probe include, e.g.,radioactive isotopes, fluoresceins or enzymes such as alkalinephosphatase.

In method (4), the detection of the protein products expressed by humanSNARE protein 25 gene can be carried out by immunology methods, such asWestern blotting, radioimmunoassay, and ELISA.

The method of amplification of DNA/RNA by PCR (Saiki, et al. Science1985; 230:1350-1354) is preferably used to obtain the polynucleotide ofthe invention. Especially when it is difficult to obtain the full-lengthcDNA, the method of RACE (RACE—cDNA terminate rapid amplification) ispreferably used. The primers used in PCR can be selected according tothe polynucleotide sequence information of the invention disclosedherein, and can be synthesized by conventional methods. The amplifiedDNA/RNA fragments can be isolated and purified by conventional methodssuch as gel electrophoresis.

Sequencing of polynucleotide sequence of the gene of the invention orits various DNA fragments can be carried out by the conventional dideoxysequencing method (Sanger et al. PNAS, 1977, 74: 5463-5467). Sequencingof polynucleotide sequence can also be carried out using thecommercially available sequencing kits. In order to obtain thefull-length cDNA sequence, it is necessary to repeat the sequencingprocess. Sometimes, it is needed to sequence the cDNA of several clonesto obtain the full-length cDNA sequence.

The invention further relates to a vector comprising the polynucleotideof the invention, a genetically engineered host cell transformed withthe vector of the invention or directly with the sequence encoding humanSNARE protein 25, and a method for producing the polypeptide of theinvention by recombinant techniques.

In the present invention, the polynucleotide sequences encoding humanSNARE protein 25 may be inserted into a vector to form a recombinantvector containing the polynucleotide of the invention. The term “vector”refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant virusor mammalian virus such as adenovirus, retrovirus or any other vehicleknown in the art. Vectors suitable for use in the present inventioninclude, but are not limited to the T7-based expression vector forexpression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), thepMSXND expression vector for expression in mammalian cells (Lee andNathans, J Biol. Chem., 263:3521, 1988) and baculovirus-derived vectorsfor expression in insect cells. Any plasmid or vector can be used toconstruct the recombinant expression vector as long as it can replicateand is stable in the host. One important feature of an expression vectoris that the expression vector typically contains an origin ofreplication, a promoter, a marker gene as well as translation regulatorycomponents.

Methods known in the art can be used to construct an expression vectorcontaining the DNA sequence of human SNARE protein 25 and appropriatetranscription/translation regulatory components. These methods includein vitro recombinant DNA technique, DNA synthesis technique, in vivorecombinant technique and so on (Sambroook, et al. Molecular Cloning, aLaboratory Manual, cold Spring Harbor Laboratory. New York, 1989). TheDNA sequence is operatively linked to a proper promoter in an expressionvector to direct the synthesis of mRNA. Exemplary promoters are lac ortrp promoter of E. coli; PL promoter of λ phage; eukaryotic promotersincluding CMV immediate early promoter, HSV thymidine kinase promoter,early and late SV40 promoter, LTRs of retrovirus, and other knownpromoters which control gene expression in the prokaryotic cells,eukaryotic cells or viruses. The expression vector may further comprisea ribosome binding site for initiating translation, transcriptionterminator and the like. Transcription in higher eukaryotes is increasedby inserting an enhancer sequence into the vector. Enhancers arecis-acting elements of DNA, usually about from 10 to 300 bp in lengththat act on a promoter to increase gene transcription level. Examplesinclude the SV40 enhancer on the late side of the replication origin 100to 270 bp, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Further, the expression vector preferably comprises one or moreselective marker genes to provide a phenotype for the selection of thetransformed host cells, e.g., the dehydrofolate reductase, neomycinresistance gene and GFP (green flurencent protein) for eukaryotic cells,as well as tetracycline or ampicillin resistance gene for E. coli.

An ordinarily skilled in the art know clearly how to select appropriatevectors, transcriptional regulatory elements, e.g., promoters,enhancers, and selective marker genes.

According to the invention, polynucleotide encoding human SNARE protein25 or recombinant vector containing said polynucleotide can betransformed or transfected into host cells to construct geneticallyengineered host cells containing said polynucleotide or said recombinantvector. The term “host cell” means prokaryote, such as bacteria; orprimary eukaryote, such as yeast; or higher eukaryotic, such asmammalian cells. Representative examples are bacterial cells, such as E.coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;plant cells; insect cells such as Drosophila S2 or Sf9; animal cellssuch as CHO, COS or Bowes melanoma.

Transformation of a host cell with the DNA sequence of invention or arecombinant vector containing said DNA sequence may be carried out byconventional techniques as are well known to those skilled in the art.When the host is prokaryotic, such as E. coli, competent cells, whichare capable of DNA uptake, can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl2 methodusing procedures well known in the art. Alternatively, MgCl2 can beused. Transformation can also be carried out by electroporation, ifdesired. When the host is an eukaryote, transfection methods as well ascalcium phosphate precipitation may be used. Conventional mechanicalprocedures such as micro-injection, electroporation, orliposome-mediated transfection may also be used.

The recombinant human SNARE protein 25 can be expressed or produced bythe conventional recombinant DNA technology (Science, 1984; 224:1431),using the polynucleotide sequence of the invention. The steps generallyinclude:

(1) transfecting or transforming the appropriate host cells with thepolynucleotide (or variant) encoding human SNARE protein 25 of theinvention or the recombinant expression vector containing saidpolynucleotide;

(2) culturing the host cells in an appropriate medium; and

(3) isolating or purifying the protein from the medium or cells.

In Step (2) above, depending on the host cells used, the medium forcultivation can be selected from various conventional mediums. The hostcells are cultured under a condition suitable for its growth until thehost cells grow to an appropriate cell density. Then, the selectedpromoter is induced by appropriate means (e.g., temperature shift orchemical induction) and cells are cultured for an additional period.

In Step (3), the recombinant polypeptide may be included in the cells,or expressed on the cell membrane, or secreted out of the cell. Ifdesired, physical, chemical and other properties can be utilized invarious isolation methods to isolate and purify the recombinant protein.These methods are well-known to those skilled in the art and include,but are not limited to conventional renaturation treatment, treatment bya protein precipitant (such as salt precipitation), centrifugation, celllysis by osmosis, sonication, supercentrifugation, molecular sievechromatography or gel chromatography, adsorption chromatography, ionexchange chromatography, HPLC, and any other liquid chromatography, anda combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to illustrate the embodiment of theinvention, not to limit the scope of invention defined by the claims.

FIG. 1 shows an alignment comparison of amino acid sequences of HumanSNARE protein 25 of the invention and SNARE-29KD in rat Golgi complex.The upper sequence is Human SNARE protein 25, and the lower sequence isSNARE-29KD in rat Golgi complex. The identical and similar amino acidsare indicated by a one-letter code of amino acid and “+” respectively.

FIG. 2 shows the SDS-PAGE of the isolated Human SNARE protein 25, whichhas a molecular weight of 25 kDa. The isolated protein band is markedwith an arrow.

EXAMPLES

The invention is further illustrated by the following examples. It isappreciated that these examples are only intended to illustrate theinvention, not to limit the scope of the invention. For the experimentalmethods in the following examples, they are performed under routineconditions, e.g., those described by Sambrook. et al., in MoleculeClone: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress, 1989, or as instructed by the manufacturers, unless otherwisespecified.

Example 1

Cloning of Human SNARE Protein 25 Gene

Total RNA from a human embryonic brain was extracted by the one-stepmethod with guanidinium isocyanate/phenol/chloroform. The poly(A) mRNAwas isolated from the total RNA with Quik mRNA Isolation Kit (Qiegene).cDNA was prepared by reverse transcription with 2 μg poly(A) mRNA. ThecDNA fragments were inserted into the polyclonal site of pBSK(+) vector(Clontech) using Smart cDNA cloning kit (Clontech) and then transformedinto DH5α to form the cDNA library. The 5′- and 3′-ends of all cloneswere sequenced with Dye terminate cycle reaction sequencing kit(Perkin-Elmer) and ABI 377 Automatic Sequencer (Perkin-Elmer). Thesequenced cDNA were compared with the public database of DNA sequences(Genebank) and the DNA sequence of one clone 1007F10 was found to be anovel DNA sequence. The inserted cDNA sequence of clone 1007F10 wasdual-directionally sequenced with a serial of synthesized primers. Itwas indicated that the full length cDNA contained in clone 1007F10 was4201 bp (SEQ ID NO: 1) with a 675 bp ORF located in positions 172-846which encoded a novel protein (SEQ ID NO: 2). This clone was namedpBS-1007F10 and the encoded protein was named Human SNARE protein 25.

Example 2

Homology Search of cDNA Clone

The homology research of the DNA sequence and its protein sequence ofHuman SNARE protein 25 of the invention were performed by Blast (Basiclocal Alignment search tool) (Altschul, S F et al. J. Mol. Biol. 1990;215: 403-10) in databases such as Genbank, Swissport, etc. The mosthomologous gene to Human SNARE protein 25 of the invention is knownSNARE-29KD in rat Golgi complex. The Genbank accession number of itsencoded protein is AF035823. The alignment result of the protein wasshown in FIG. 1. Two proteins are highly homologous with an identity of91% and a similarity of 93%.

Example 3

Cloning Human SNARE Protein 25 Gene by RT-PCR

The template was total RNA extracted from a human embryonic brain. Thereverse transcription was carried out with oligo-dT primer to producecDNAs. After cDNA purified with Qiagen Kit, PCR was carried out with thefollowing primers:

Primer 1: 5′-GGAGATTGCGACGAACAACCAGG-3′ (SEQ ID NO: 3)

Primer 2: 5′-CCTTAACAGTTGTTTAATGCAATT-3′ (SEQ ID NO: 4)

Primer 1 is the forward sequence started from position 1 of 5′ end ofSEQ ID NO: 1.

Primer 2 is the reverse sequence of the 3′ end of SEQ ID NO: 1.

The amplification condition was a 50 ul reaction system containing 50mmol/L KCl, 10 mmol/L Tris-Cl (pH8. 5), 1.5 mmol/L MgCl₂, 200 umol/LdNTP, 10 pmol of each primer, 1U Taq DNA polymerase(Clontech). Thereaction was performed on a PE 9600 DNA amplifier with the followingparameters: 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 min for 25cycles. β-actin was used as a positive control, and a blank template, asa negative control in RT-PCR. The amplified products were purified witha QIAGEN kit, and linked with a pCR vector (Invitrogen) using a TACloning Kit. DNA sequencing results show that the DNA sequence of PCRproducts was identical to nucleotides 1-4201 bp of SEQ ID NO: 1.

Example 4

Northern Blotting of Expression of Human SNARE Protein 25 Gene

Total RNA was extracted by one-step method (Anal. Biochem 1987, 162,156-159) with guanidinium isocyanate-phenol-chloroform. That is,homogenate the organize using 4M guanidinium isocyanate-25 mM sodiumcitrate, 0.2 sodium acetate(pH4.0), add 1 volume phenol and 1/5 volumechloroform-isoamyl alcohol(49:1), centrifuge after mixing. Take out thewater phase, add 0.8 volume isopropyl alcohol, then centrifuge themixture. Wash the RNA precipitation using 70% ethanol, then dry, thendissolve it in the water. 20μg RNA was electrophoresed on the 1.2%agarose gel containing 20 mM 3-(N-morpholino) propane sulfonic acid(pH7.0)-5 mM sodium acetate-imM EDTA- 2. 2M formaldehyde. Then transfer itto a nitrocellulose filter. Prepare the ³²p-labelled DNA probe withα-³²P dATP by random primer method. The used DNA probe is the codingsequence (172 bp-846 bp) of Human SNARE protein 25 amplified by PCRindicated in FIG. 1. The nitrocellulose filter with the transferred RNAwas hybridized with the ³²P-labelled DNA probe (2×10⁶cpm/ml) overnightin a buffer containing 50% formamide-25 mM KH₂PO₄(Ph7.4)-5× Denhardt'ssolution and 200 μg /ml salmine. Then wash the filter in the 1×SSC-0.1%SDS, at 55° C., for 30 min. Then analyze and quantitative determinateusing Phosphor Imager.

Example 5

In vitro Expression, Isolation and Purification of Recombinant HumanSNARE Protein 25

A pair of primers for specific amplification was designed based on SEQID NO: 1 and the encoding region in FIG. 1, the sequences are asfollows:

Primer3: 5′-CCCCATATGATGTGTCGTCCGACTTCGAAGGTTACG-3′ (SEQ ID NO: 5)

Primer4: 5′-CCCGTCGACTCAGTGTCTTCTGACAGAAAAAGTG-3′ (SEQ ID NO: 6)

These two primers contain a NdeI and SalI cleavage site on the 5′ endrespectively. Within the sites are the coding sequences of the 5′ and 3′end of the desired gene. NdeI and SalI cleavage sites were correspondingto the selective cleavage sites on the expression vector pET-28b(+)(Novagen, Cat. No. 69865.3). PCR amplification was performed with theplasmid pBS-1007F10 containing the full-length target gene as atemplate. The PCR reaction was subject to a 50 μl system containing 10pg pBS-1007F10plasmid, 10 pmol of Primer-3 and 10 pmol of Primer-4, 1 μlof Advantage polymerase Mix (Clontech). The parameters of PCR were 94°C. 20 sec, 60° C. 30 sec, and 68° C. 2 min for 25 cycles. Afterdigesting the amplification products and the plasmid pET-28(+) by NdeIand SalI, the large fragments were recovered and ligated with T4 ligase.The ligated product was transformed into E. coli DH5α with the calciumchloride method. After cultured overnight on a LB plate containing afinal concentration of 30 μg/ml kanamycin, positive clones were selectedout using colony PCR and then sequenced. The positive clone(pET-1007F10) with the correct sequence was selected out and therecombinant plasmid thereof was transformed into BL21(DE3)plySs(Novagen) using the calcium chloride method. In a LB liquid mediumcontaining a final concentration of 30 μg/ml of kanamycin, the hostbacteria BL21(pET-1007F10) were cultured at 37° C. to the exponentialgrowth phase, then IPTG were added with the final concentration of 1mmol/L, the cells were cultured for another 5 hours, and thencentrifuged to harvest the bacteria. After the bacteria were sonicated,the supernatant was collected by centrifugation. Then the purifieddesired protein—Human SNARE protein 25 was obtained by a His. Bind QuickCartridge (Novagen) affinity column with binding 6His-Tag. SDS-PAGEshowed a single band at 25 kDa (FIG. 2). The band was transferred ontothe PVDF membrane and the N terminal amino acid was sequenced by EdamsHydrolysis, which shows that the first 15 amino acids on N-terminus wereidentical to those in SEQ ID NO: 2.

Example 6

Preparation of Antibody Against Human SNARE Protein 25

The following specific Human SNARE protein 25 polypeptide wassynthesized by a polypeptide synthesizer (PE-ABI):NH2-Met-Ser-Ser-Asp-Phe-Glu-Gly-Tyr-Glu-Gln-Asp-Phe-Ala-Val-Leu-COOH(SEQ ID NO: 7). The polypeptide was conjugated with hemocyanin andbovine serum albumin (BSA) respectively to form two composites (SeeAvrameas et al., Immunochemistry, 1969, 6:43). 4 mg ofhemocyanin-polypeptide composite was used to immunize rabbit togetherwith Freund's complete adjuvant. The rabbit was re-immunized with thehemocyanin-polypeptide composite and Freund's incomplete adjuvent 15days later. The titer of antibody in the rabbit sera was determined witha titration plate coated with 15 μg/ml BSA-polypeptide composite byELISA. The total IgG was isolated from the sera of an antibody positiverabbit with Protein A-Sepharose. The polypeptide was bound to Sepharose4B column activated by cyanogen bromide. The antibodies against thepolypeptide were isolated from the total IgG by affinity chromatography.The immunoprecipitation approved that the purified antibodies couldspecifically bind to Human SNARE protein 25.

INDUSTRIAL APPLICABILITY

The polypeptide of the invention and antagonists, agonists andinhibitors thereof can be directly used for the treatment of diseases,e.g., various malignant tumors or cancers, dermatitis, inflammation, HIVinfection and immune system diseases.

The polypeptide of this invention, human SNARE protein 25, is a SNAP(soluble NSF-binding protein) receptor protein. Its functions in cellsinclude: (1) specific recognition between vesicle and target-membrane;and (2) catalyzing the fusion of lipid bi-layers. Moreover, thepolypeptide of the invention has a significant effect on the processesof secretion of platelet granules, release of neurotransmitter and soon. So as the receptor of NSF binding-protein, SNARE is an importantprotein in the above processes.

The polypeptide of the invention (Human SNARE protein 25) is of greatsignificance in the processes of secretion of macro-molecules (e.g.protein), cellular transportation, effects of hormones and other drugmolecules on cells, metabolism regulation, cellular recognition, andimmunization, etc.

The polypeptide of the invention (human SNARE protein 25) can be used todiagnose and treat many diseases, including but not limited: variousmalignant tumors, endocrine system diseases, diseases related to growthand development, nervous system diseases, immune diseases, acquiredimmune deficiency symptoms (AIDS).

The polypeptide also can be used to treat common human nervous systemdiseases, including:

(1) Brain Diseases: cerebrovascular diseases: transient cerebralischemia, cerebral embolism, encephalorrhagia, subarachnoid hemorrhage;Intracranial space-occupying lesion: neuroglioma, meningioma,neurofibroma, pituitary adenoma, and encephalic granuloma.

Neurodegenerative Diseases: Alzheimer's Disease, Parkinson's Disease,chorea, depression, amnesia, Huntington's disease, epilepsy, migraine,dementia, multiple sclerosis.

(2) Neuromuscular Diseases: myasthenia gravis, spinal muscle atrophy,muscle pseudohypertrophy, Duchenne muscle cacotrophia, tetanic musclecacotrophia, muscle catatonia tardive dyskinesia, muscle tensionhandicap.

(3) Neurocataneous Syndrome: fibroneuroma, tubercular sclerosis,cerebral trigeminal angioma, ataxia-telangiectasia.

(4) Mental Diseases: schizophrenia, depression, paranoia, anxiety,compulsion, fearing, and neurasthenia.

(5) Spinal Cord Diseases: acute myelitis, spinal cord compression

(6) Peripheral Nervous Diseases: prosopalgia, facial palsy, bulbarpalsy, sciatica, and Guillain-Barre Syndrome.

The polypeptide of the invention may also be used to treat developmentaldisorders, including: bifid spine, cranioschisis, anencephaly,craniocele, schizencephalic porencephaly, Down's syndrome, congenitalencephaledema, aqueduct cacogenesis, achondroplastic dwarf, hypogenesisof spinal skeleton, pseudocartilage hypoplasia, Langer-Giedion syndrome,funnel chest, hypogenitalism, congenital adrenal cortical hyperplasia,epispadias, enorchia, cacogenesis syndrome associated with microsoma(e.g. Conradi syndrome and Danbolt-closs syndrome), congenital cataract,congenital phacometachoresis; congenital microblepharia, retinadysplasia, congenital optic atrophy, congenital sensory nerve hearingloss, acrorhagadia, teratism, williams syndrome, Alagille syndrome,Bechwith-Widedemann syndrome and so on.

Human SNARE protein 25 may also be used to treat various tumors, suchas: tumors of epithelial tissues (e.g. basal lamina epithelial,imbrication epithelial, mucus cell etc.), connective tissues (e.g. Fibertissue, adipose tissue, cartilage tissue, smooth muscle tissue, bloodvessel and lymphangioencothelium tissue, etc.), hemopoietic tissues(e.g. B cell, T cell, tissue cell, etc), central nervous tissues,peripheral nervous tissues, endorcrine tissues, sexual gland tissues,special tissues (e.g. tooth tissue). For example: gastric cancinoma,hepatoma, large intestines cancer, galactophore cancer, lung cancer,prostate cancer, cancer of uterine cervix, pancreatic cancer, oesophaguscancer, etc.

The polypeptide of the invention (human SNARE protein 25) also is animmunomodulator, functioning either as an immuno-depressor orimmuno-stimulant. It can be used to treat abnormal immunoreaction,anergy and immune tolerance, and host-defense failure. The polypeptideand its antibody may also be effective in treating diseases caused bydemages to the immune system, immune deficiency or disorders, such ashemopoietic system diseases (e.g. pernicious anemia), dermatitis (e.g.psoriasis), autoimmune diseases (e.g. rheumatoid arthritis), radioactivediseases and the formation and regulation lymphocytes.

The polypeptide (human SNARE protein 25) are used for treatinginflammation reactions, such as: allergic reaction, bronchial asthma,allergic pneumonia, adult respiratory distress, lung eosinopenia,sarcoidosis, rheumatic arthritis, rheumatoid arthritis, osteoarthritis,cholecystitis, glomerulonephritis, immune complex-mediatedglomerulonephritis, acute uveitis, osteoporosis, dermatomyositis, hives,specific dermatitis, hemochromatiosis, polymysitis, Addision disease,Greffis disease, chronic active hepatitis, Green-Barry syndrome,encephalic granuloma, Wegener granuloma, autoimmune thyroiditis,autoimmune interstitial nephritis, ulcerative colitis, hemophthisis,panctratitits, segmental ileitis, myocarditis, atherosclerosisatherosis, multiple scleroderma, inflammation caused by infection andinjury and so on.

The invention also provides methods for screening compounds so as toidentify an agent which enhances Human SNARE protein 25 activity(agonists) or decrease Human SNARE protein 25 activity (antagonists).The agonists enhance the biological functions of Human SNARE protein 25such as inactivation of cell proliferation, while the antagonistsprevent and alleviate the disorders associated with the excess cellproliferation, such as various cancers. For example, in the presence ofan agent, the mammal cells or the membrane preparation expressing HumanSNARE protein 25 can be incubated with the labeled Human SNARE protein25 to determine the ability of the agent to enhance or repress theinteraction.

Antagonists of Human SNARE protein 25 include antibodies, compounds,receptor deletants and analogues. The antagonists of Human SNARE protein25 can bind to Human SNARE protein 25 and eliminate or reduce itsfunction, or inhibit the production of Human SNARE protein 25, or bindto the active site of said polypeptide so that the polypeptide can notfunction biologically.

When screening for compounds as an antagonist, Human SNARE protein 25may be added into a biological assay. It can be determined whether thecompound is an antagonist or not by determining its effect on theinteraction between Human SNARE protein 25 and its receptor. Using thesame method as that for screening compounds, receptor deletants andanalogues acting as antagonists can be selected. Polypeptide moleculescapable of binding to Human SNARE protein 25 can be obtained byscreening a polypeptide library comprising various combinations of aminoacids bound onto a solid matrix. Usually, Human SNARE protein 25 islabeled in the screening.

The invention further provides a method for producing antibodies usingthe polypeptide, and its fragment, derivative, analogue or cells as anantigen. These antibodies may be polyclonal or monoclonal antibodies.The invention also provides antibodies against epitopes of Human SNAREprotein 25. These antibodies include, but are not limited to, polyclonalantibody, monoclonal antibody, chimeric antibody, single-chain antibody,Fab fragment and the fragments produced by a Fab expression library.

Polyclonal antibodies can be prepared by immunizing animals, such asrabbit, mouse, and rat, with Human SNARE protein 25. Various adjuvants,including but are not limited to Freund's adjuvant, can be used toenhance the immunization. The techniques for producing Human SNAREprotein 25 monoclonal antibodies include, but are not limited to, thehybridoma technique (Kohler and Milstein. Nature, 1975, 256: 495-497),the trioma technique, the human B-cell hybridoma technique, theEBV-hybridoma technique and so on. A chimeric antibody comprising aconstant region of human origin and a variable region of non-humanorigin can be produced using methods well-known in the art (Morrison etal, PNAS, 1985, 81: 6851). Furthermore, techniques for producing asingle-chain antibody (U.S. Pat. No. 4,946,778) are also useful forpreparing single-chain antibodies against Human SNARE protein 25.

The antibody against Human SNARE protein 25 can be used inimmunohistochemical method to detect the presence of Human SNARE protein25 in a biopsy specimen.

The monoclonal antibody specific to Human SNARE protein 25 can belabeled by radioactive isotopes, and injected into human body to tracethe location and distribution of Human SNARE protein 25. Thisradioactively labeled antibody can be used in the non-woundingdiagnostic method for the determination of tumor location andmetastasis.

Antibodies can also be designed as an immunotoxin targeting a particularsite in the body. For example, a monoclonal antibody having highaffinity to Human SNARE protein 25 can be covalently bound to bacterialor plant toxins, such as diphtheria toxin, ricin, ormosine. One commonmethod is to challenge the amino group on the antibody with sulfydrylcross-linking agents, such as SPDP, and bind the toxin onto the antibodyby interchanging the disulfide bonds. This hybrid antibody can be usedto kill Human SNARE protein 25-positive cells.

The antibody of the invention is useful for the therapy or theprophylaxis of disorders related to the Human SNARE protein 25. Theappropriate amount of antibody can be administrated to stimulate orblock the production or activity of Human SNARE protein 25.

The invention further provides diagnostic assays for quantitative and insitu measurement of Human SNARE protein 25 level. These assays are wellknown in the art and include FISH assay and radioimmunoassay. The levelof Human SNARE protein 25 detected in the assay can be used toillustrate the importance of Human SNARE protein 25 in diseases and todetermine the diseases associated with Human SNARE protein 25.

The polypeptide of the invention is useful in the analysis ofpolypeptide profile. For example, the polypeptide can be specificallydigested by physical, chemical, or enzymatic means, and then analyzed byone, two or three dimensional gel electrophoresis, preferably byspectrometry.

New Human SNARE protein 25 polynucleotides also have many therapeuticapplications. Gene therapy technology can be used in the therapy ofabnormal cell proliferation, development or metabolism, which are causedby the loss of Human SNARE protein 25 expression or the abnormal ornon-active expression of Human SNARE protein 25. Recombinant genetherapy vectors, such as virus vectors, can be designed to expressmutated Human SNARE protein 25 so as to inhibit the activity ofendogenous Human SNARE protein 25. For example, one form of mutatedHuman SNARE protein 25 is a truncated Human SNARE protein 25 whosesignal transduction domain is deleted. Therefore, this mutated HumanSNARE protein 25 can bind the downstream substrate without the activityof signal transduction. Thus, the recombinant gene therapy vectors canbe used to treat diseases caused by abnormal expression or activity ofHuman SNARE protein 25. The expression vectors derived from a virus,such as retrovirus, adenovirus, adeno-associated virus, herpes simplexvirus, parvovirus, and so on, can be used to introduce the Human SNAREprotein 25 gene into the cells. The methods for constructing arecombinant virus vector harboring Human SNARE protein 25 gene aredescribed in the literature (Sambrook, et al. supra). In addition, therecombinant Human SNARE protein 25 gene can be packed into liposome andthen transferred into the cells.

The methods for introducing the polynucleotides into tissues or cellsinclude directly injecting the polynucleotides into tissue in the body;or introducing the polynucleotides into cells in vitro with vectors,such as virus, phage, or plasmid, etc, and then transplanting the cellsinto the body.

Also included in the invention are ribozyme and the oligonucleotides,including antisense RNA and DNA, which inhibit the translation of theHuman SNARE protein 25 mRNA. Ribozyme is an enzyme-like RNA moleculecapable of specifically cutting certain RNA. The mechanism is nucleicacid endo-cleavage following specific hybridization of ribozyme moleculeand the complementary target RNA. Antisense RNA and DNA as well asribozyme can be prepared by using any conventional techniques for RNAand DNA synthesis, e.g., the widely used solid phase phosphite chemicalmethod for oligonucleotide synthesis. Antisense RNA molecule can beobtained by the in vivo or in vitro transcription of the DNA sequenceencoding said RNA, wherein said DNA sequence is integrated into thevector and downstream of the RNA polymerase promoter. In order toincrease its stability, a nucleic acid molecule can be modified in manymanners, e.g., increasing the length of two the flanking sequences,replacing the phosphodiester bond with the phosphothioester bond in theoligonucleotide.

The polynucleotide encoding Human SNARE protein 25 can be used in thediagnosis of Human SNARE protein 25 related diseases. The polynucleotideencoding Human SNARE protein 25 can be used to detect whether HumanSNARE protein 25 is expressed or not, and whether the expression ofHuman SNARE protein 25 is normal or abnormal in the case of diseases.For example, Human SNARE protein 25 DNA sequences can be used in thehybridization with biopsy samples to determine the expression of HumanSNARE protein 25. The hybridization methods include Southern blotting,Northern blotting and in situ blotting, etc., which are well-known andestablished techniques. The corresponding kits are commerciallyavailable. A part of or all of the polynucleotides of the invention canbe used as probe and fixed on a microarray or DNA chip for analysis ofdifferential expression of genes in tissues and for the diagnosis ofgenes. The Human SNARE protein 25 specific primers can be used inRNA-polymerase chain reaction and in vitro amplification to detecttranscripts of Human SNARE protein 25.

Further, detection of mutations in Human SNARE protein 25 gene is usefulfor the diagnosis of Human SNARE protein 25-related diseases. Mutationsof Human SNARE protein 25 include site mutation, translocation,deletion, rearrangement and any other mutations compared with thewild-type Human SNARE protein 25 DNA sequence. The conventional methods,such as Southern blotting, DNA sequencing, PCR and in situ blotting, canbe used to detect a mutation. Moreover, mutations sometimes affects theexpression of protein. Therefore, Northern blotting and Western blottingcan be used to indirectly determine whether the gene is mutated or not.

Sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.There is a current need for identifying particular sites of gene on thechromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphism) are presently available for markingchromosomal location. The mapping of DNA to chromosomes according to thepresent invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-35 bp) from the cDNA. These primers are then used for PCRscreening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the oligonucleotideprimers of the invention, sublocalization can be achieved with panels offragments from specific chromosomes or pools of large genomic clones inan analogous manner. Other mapping strategies that can similarly be usedto map to its chromosome include in situ hybridization, prescreeningwith labeled flow-sorted chromosomes and preselection by hybridizationto construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. For a review of this technique, seeVerma et al., Human Chromosomes: a Manual of Basic Techniques, PergamonPress, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis.

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be the causeof the disease. Comparison of affected and unaffected individualsgenerally involves first looking for structural alterations in thechromosomes, such as deletions or translocations, that are visible fromchromosome level, or detectable using PCR based on that DNA sequence.With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50 to 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

According to the invention, the polypeptides, polynucleotides and itsmimetics, agonists, antagonists and inhibitors may be employed incombination with a suitable pharmaceutical carrier. Such a carrierincludes but is not limited to water, glucose, ethanol, salt, buffer,glycerol, and combinations thereof. Such compositions comprise a safeand effective amount of the polypeptide or antagonist, as well as apharmaceutically acceptable carrier or excipient with no influence onthe effect of the drug. These compositions can be used as drugs indisease treatment.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. With such container(s)there may be a notice from a governmental agency, that regulates themanufacture, use or sale of pharmaceuticals or biological products, thenotice reflects government's approval for the manufacture, use or salefor human administration. In addition, the polypeptides of the inventionmay be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner, such as through topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. HumanSNARE protein 25 is administered in an amount, which is effective fortreating and/or prophylaxis of the specific indication. The amount ofHuman SNARE protein 25 administrated on patient will depend upon variousfactors, such as delivery methods, the subject health, judgment of theskilled clinician.

1. An isolated polypeptide having a human SNARE protein 25 activity andcomprising an amino acid sequence of SEQ ID NO:
 2. 2. An isolatedpolypeptide having a human SNARE protein 25 activity and comprising anamino acid sequence that is at least 97% identical to SEQ ID NO:
 2. 3. Apharmaceutical composition comprising a polypeptide according to claim2, and a pharmaceutically acceptable carrier.